The completion of the human genome project has greatly increased the need for an efficient method for providing a large quantity of genetic information necessary for genetic disease diagnosis, treatment, and prevention. Despite the development of DNA polymerase chain reaction (PCR) and its automization, Sanger's method for nucleotide sequencing is still burdensome and delicate to handle, and needs lots of time, effort, and cost. Thus, there have been many attempts to find a new nucleotide sequencing system capable of analyzing a large number of genes.
A DNA chip refers to a microarray of less than 1 square inch, which is formed by immobilizing oligonucleotide probes, each probe including a few to hundreds of nucleotides, at hundreds to hundreds of thousands of appropriate positions on a solid surface made of silicon, surface-modified glass, polypropylene, or activated polyacrylamide. As a target DNA fragment is bound to the DNA chip, the target DNA fragment complementarily hybridizes to the oligonucleotide probes immobilized on the DNA chip. The hybridization is optically or radiochemically observed and analyzed to identify the nucleotide sequence of the target DNA.
Use of the DNA chip reduces the size of a DNA assay system and enables genetic assay with a trace of a sample. In addition, multiple sequences of a target DNA can be simultaneously assayed, thereby conveniently and rapidly providing the genetic information of the target DNA at low costs. The DNA chip can assay a large amount of genetic information and the relevancy of the genes within a short period of time. Accordingly, the DNA chip is expected to have wide applications, for example, to genetic disorder and cancer diagnosis, mutant and pathogen detection, gene expression assay, drug discovery, etc. In addition, the DNA chip can be used as a microorganism or pollutant detector to find out antidotal genes and further to produce antidotes on a large scale based on genetic recombination technologies. The DNA chip can lead to great improvements in most biological industries, including the production of medicinal crops or low-fat meat.
DNA chips are classified into an oligo-chip and a cDNA chip according to the type of probes immobilized thereon. According to the manufacturing method, DNA chips are classified into a lithography chip, a pin-type spotting chip, an ink-jet type spotting chip, and an electronic addressing DNA chip that electronically integrates DNA into a substrate.
The first generation DNA chip, a 2-dimensional (2D) chip in which oligonucleotides are attached to a substrate as a monolayer, causes a considerable error when the degree of hybridization is relatively compared, due to spot-to-spot variation of the probes attached to the substrate, and has low sensitivity, and thus needs an expensive confocal fluorescence microscope to detect the hybridized DNA. In addition, the surface of the solid substrate is treated with a mixture of an organic solvent and an aqueous solution to induce chemical reactions.
U.S. Pat. No. 5,744,305 discloses an example of the first generation DNA chip manufactured by photolithography using a photolabile protecting group. In the first generation DNA chip, various polymers such as peptides and oligonucleotides are arrayed on the substrate.
To address the drawbacks of the first generation DNA chip, the following second generation DNA chip was developed. U.S. Pat. Nos. 5,736,257 and 5,847,019 disclose methods for manufacturing biochips, in which a hydroxyl (OH) group on a substrate is treated with silane to form a molecular layer of a vinyl group on the substrate, and a network layer is formed on the molecular layer through polymerization of acrylamide and activated and patterned using light to bind the network layer to a biomaterial. In these methods, due to a 3-D gel network of the polyacrylamide formed on the molecular layer of the vinyl group, a quantitative variation of probes immobilized thereon is reduced, and chip sensitivity is improved. However, disadvantageously, the cost is high and a long assay period of time is required.
U.S. Pat. Nos. 5,552,270, 5,741,700, and 5,770,721 disclose DNA sequence assay methods using a second generation DNA chip including an oligonucleotide array matrix and a solid substrate. The matrix is bound to the solid substrate via a gel layer in a pattern of square dots spaced a constant distance from one another. In the manufacture of the DNA chip, a polyacrylamide gel is applied between two glass slides spaced about 30 μm apart. One glass slide having the size of 100×100×30 μm3 is removed, and the remaining glass slide coated with the gel is dried and formed into a dot pattern by partially removing the gel through a mechanical tool or laser. The amide group of the gel layer is chemically transformed into reactive hydrazide form, and DNA probes having N-methyluridine at 5′-terminal are transformed into dialdehyde. The oligonucleotide probe is immobilized on the substrate in three dimensions by reacting the probe with the reactive gel.
The DNA chip is manufactured through multiple processes over a longer period of time of from 1 to 2 days, wherein each of the processes is followed by a thorough wash process. Accordingly, the assay yield is susceptible to reaction conditions, and irregular adsorption of the target DNA to the gel surface increases background noise.
WO 00/65097 and WO 00/2899 disclose 3-D DNA chips using a hydrogel having an isocyanate (NCO) group. The hydrogel is a hydrophilic network polymer which is glassy in a dehydrated state and swells to form a gel in the presence of water. According to the disclosures, the isocyanate group is known to be involved in covalent bonding between the hydrogel and probes as well as in polymerization of the hydrogel itself. A hydrogel having the isocyanate group, for example, polyethyleneoxide having an isocyanate group or a copolymer of polyethyleneoxide having an isocyanate group and polypropyleneoxide, and probes having an amino group at the 5′-terminal are mixed in an aqueous solution, and the probes are immobilized on the gel in three dimensions and reacted with an amino group on the glass surface to attach the probes on the substrate.
The isocyanate group is hydrolyzed too rapidly to control. To prevent the hydrolysis of the isocyanate group there is a need to manufacture a DNA chip at low temperature. As the isocyanate groups that neither covalently bind to the probes nor are involved in gel polymerization are hydrolyzed, the polymerization is accelerated to increase viscosity and to solidify a spotting pin. Also, generation of carbon dioxide makes spotting size control difficult.